The present invention is related to the systems and methods for characterizing the molecular make-up an unknown sample, and more particularly to systems and methods for detecting and identifying molecular events in a sample using a resonant test structure.
Virtually every area of biological science is in need of a system to determine the ability of molecules of interest to interact with other molecules. Likewise, the ability to detect the presence and/or physical and functional properties of biological molecules on a small scale is highly desirable. Such molecular interactions, as well as the detection of functional and physical properties of molecules, are referred to here as molecular events. The need to detect molecular events ranges from the basic science research lab, where chemical messenger pathways are being mapped out and their functions correlated to disease processes, to pre-clinical investigations, where candidate drugs are being evaluated for potential in vivo effectiveness. The need to detect physical and functional properties is also present in these research areas, such as for functional analysis of a newly discovered protein or of a genetic (or synthetic) variant of a molecule of know biological importance. Other areas that benefit from a better understanding of molecular events include pharmaceutical research, military applications, veterinary, food, and environmental applications. In all of these cases, knowledge of the ability of a particular analyte to bind a target molecule is highly useful, as is information relating to the quality of that binding (e.g., affinity and on-off rate), and other functional information about new molecules, particularly when information can be obtained from a small amount of sample.
Numerous methodologies have been developed over the years in attempts to meet the demands of these fields, such as Enzyme-Linked Immunosorbent Assays (ELISA), Radio-Immunoassays (RIA), numerous fluorescence assays, nuclear magnetic resonance (NMR) spectroscopy, and calorimetric assays, as well as a host of more specialized assays. Most of these assay techniques require specialized preparation, purification, or amplification of the sample to be tested. To detect a binding event between a ligand and an antiligand, for example, a detectable signal is required that signals the existence or extension of binding. Usually the signal has been provided by a label that is attached to either the ligand or antiligand of interest. Physical or chemical effects which produce detectable signals, and for which suitable labels exist, include radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity, to name a few. The label can then be detected by spectrophotometric, radiometric, or optical tracking methods.
Unfortunately, in many cases it is difficult or even impossible to label one or all of the molecules needed for a particular assay. The presence of a label also can make the molecular recognition between two molecules not function in its normal manner for many reasons, including steric effects. In addition, none of these labeling approaches determines the exact nature of the binding event, so that, for example, active-site binding to a receptor is indistinguishable from non-active-site binding, such as allosteric binding, and thus no functional information is obtained via the present detection methodologies. In general, limitations also exist in the areas of specificity and sensitivity of most assay systems. Cellular debris and non-specific binding often cause an assay to be noisy and make it difficult or impossible to extract useful information. As mentioned above, some systems are too complicated to allow the attachment of labels to all analytes of interest or to allow an accurate optical measurement to be performed. Therefore, a practical, economic, and universal detection technique that can directly monitor without a label, in real time, the presence of analytes, for instance, the extent, function and type of binding events that are actually taking place in a given system would represent a significant breakthrough.
In particular, the biomedical industry needs an improved general platform technology that has very broad applicability to a variety of water-based or other fluid-based physiological systems, such as nucleic acid binding, protein-protein interactions, and small molecule binding, as well as other compounds of interest. Ideally, the assay should not require highly specific probes, such as specific antibodies or exactly complementary nucleic acid probes. It should be able to work in native environments, such as whole blood or cytosolic mixtures, as well as other naturally occurring systems. It should operate by measuring the native properties of the molecules and not require additional labels or tracers to actually monitor the binding event. For some uses it should be able to provide information on the nature of the binding event, such as whether or not a given compound binds to the active site as an agonist or an antagonist on a particular drug receptor or if the given compound binds to an allosteric site, and not function simply as a marker to indicate whether or not the binding event has taken place. For many applications, it should be highly miniaturizable and highly parallel, so that complex biochemical pathways can be mapped out, or so that extremely small and numerous quantities of combinatorial compounds can be used in drug screening protocols. In many applications, it should further be able to monitor in real time a complex series of reactions, so that accurate kinetics and affinity information can be obtained almost immediately. Perhaps most importantly, for most commercial applications it should be inexpensive and easy to use, with few sample preparation steps, affordable electronics and disposable components, such as surface chips for bioassays that can be used for an assay and then thrown away, and it should be highly adaptable to a wide range of assay applications.
Accordingly, there exists a need for development of methods of detecting molecular events that do not require labels such as fluorophores or radioisotopes, that are quantitative and qualitative, that are specific to the molecule of interest, that are highly sensitive, and that are relatively simple to implement. The present invention fulfills many of the needs discussed above and others as well, as described herein.
The present invention provides methods and systems for detecting and identifying molecular events in a test sample without the use of labels. The method includes providing a resonant test structure having a resonant response associated therewith. A first resonant response of the resonant test structure is obtained when the resonant test structure is electromagnetically coupled to a reference sample, the reference sample having a known composition. A second resonant response is also obtained when the resonant test structure is electromagnetically coupled to the test sample having unknown composition. Subsequently, one or more first electrical parameters (the q-factor of the resonant test structure in one embodiment) are derived from the first resonant response. One or more second electrical parameters are similarly derived from the second resonant response. The similarity or difference between the first and second electrical parameters are analyzed to determine the presence or absence of the molecular event in the test sample.
The nature and advantages of the present invention will be better understood with reference to the following drawings and detailed description.